CN118059244A - Soluble Vasorin (sVASN) endogenous functional receptor - Google Patents

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a soluble Vasorin (sVASN) endogenous function receptor. In particular, the invention provides the use of a combination of one or more of the following agents in the preparation of a medicament for cancer: 1) an inhibitor of CD71, 2) an inhibitor of sVASN, or 3) an agent that blocks the binding of sVASN to its receptor CD 71; specifically, the sVASN and its receptor CD71 binding site are at amino acids 1-85 of sVASN.

Description

An endogenous functional receptor for soluble vasorin (sVASN)

Technical Field

The invention belongs to the field of biomedicine, and specifically relates to an endogenous functional receptor of soluble Vasorin (sVASN).

Background technique

VASN was first discovered and reported as a TGF-β binding protein in 2004. It is mainly expressed in the aorta, the vascular-rich placenta, and the kidneys. It can regulate the aortic injury response during the repair of vascular damage by participating in the TGF-β pathway. VASN is highly expressed in breast cancer cells, gliomas, and liver cancer cells. VASN is also contained in the exosomes of urine of patients with early IgA nephropathy. VASN is highly expressed in human brain tumor tissues. When siRNA knocks down the expression level of VASN in gliomas, the apoptosis rate induced by hypoxia or TNF-α is significantly increased. This suggests that VASN may have the effect of resisting hypoxia-induced apoptosis and promoting tumor progression, which may be one of the mechanisms by which hypoxia promotes the development of brain tumors.

Human VASN protein is a typical type I transmembrane glycoprotein with a total length of 673 amino acids and a relative molecular weight of about 72 kD predicted by bioinformatics. Its extracellular domain can be cleaved and shed by disintegrin and metalloprotease 17 (ADAM17) to become soluble VASN (sVASN). sVASN contains important functional domains such as tandem leucine-rich (LRR) domains, epidermal growth factor (EGF)-like domains and fibronectin III (FN3) domains, which are the structural basis of its biological function.

Most of the current research on VASN is based on dependence on TGF-β. VASN can selectively and non-covalently interact with TGF-β. TGF-β, also known as transforming growth factor β, is a multifunctional peptide that controls the proliferation, differentiation and other functions of many cell types. In general, VASN can act as an inhibitor of TGF-β signaling. Previous studies have found that blocking the TGF-β signaling pathway cannot completely block the effect of VASN on cells, suggesting the involvement of other mechanisms.

CD71, also known as Transferrin Receptor 1 (TfR1), interacts with transferrin (Tf) to achieve the binding of transferrin to iron. CD71 is a glycoprotein expressed on the cell surface, composed of two homodimeric subunits connected by disulfide bonds. CD71 contains 760 amino acids, including a short N-terminal intracellular domain (amino acids 1 to 67), a hydrophobic transmembrane domain (amino acids 68 to 88), and a large globular extracellular domain (amino acids 89 to 760) at the C-terminus containing a transferrin binding site. CD71 is the main receptor responsible for regulating the cellular uptake of iron in transferrin and is usually highly expressed in proliferating cells and cancer cells in the pancreas, colon, lung, breast, bladder, and lymphoid organs.

Summary of the invention

From the perspective of VASN structure, its N-segment 1-575 amino acids are mainly extracellular, and the C-terminal 597-673 amino acids are located intracellularly. After ADAM17 cleavage, its main functional domains are released to the extracellular space. So what physiological functions does the extracellularly released sVASN have? Through the EMSA combined with MS method, we found that the CD71 molecule on the surface of liver cancer cells can bind to VASN, and further IP experiments confirmed the interaction between the two molecules.

The present invention determines the specific binding of the N-terminal 1-85 amino acids of sVASN with CD71 by co-immunoprecipitation (co-IP) combined with molecular dynamics simulation; determines the binding of sVASN with CD71 by a biomacromolecule interaction instrument (BIAcore) and an enzyme-linked immunosorbent assay (ELISA) and determines the affinity to the nM level; determines that exogenous sVASN can enter endothelial cells, epidermal cells and tumor cells by Western blot and laser confocal microscopy, and the amount of exogenous sVASN entering the cells is reduced after blocking or inhibiting CD71; the culture supernatant of VASN high-expressing cells HepG2 can promote the proliferation of tumor cells T98G and U118, and promote the proliferation of human brain microvascular endothelial cells hEMEC/D3, but has no effect on the proliferation of CD71 low-expressing cells U87; by blocking or inhibiting CD71 on the cell surface, cell migration, cell scratch healing and angiogenesis caused by the endocytosis of exogenous sVASN into cells can be inhibited, providing a new method for inhibiting tumor progression, i.e., blocking or inhibiting the receptor CD71 of sVASN. The above results indicate that CD71 is a new functional receptor of sVASN.

Specifically, the present invention provides the following technical solutions:

application

In one aspect, the present invention provides use of inhibitors of CD71 in the treatment of cancer.

In another aspect, the present invention provides use of an inhibitor of sVASN in treating cancer.

In another aspect, the present invention provides use of an agent that blocks the binding of sVASN to its receptor CD71 in the treatment of cancer.

In another aspect, the present invention provides a composition and its use in treating cancer, wherein the composition comprises at least two of the following:

1) CD71 inhibitors,

2) sVASN inhibitors, or

3) Agents that block the binding of sVASN to its receptor CD71.

The "agents that block the binding of sVASN to its receptor CD71" of the present invention include but are not limited to inhibitors of CD71 and inhibitors of sVASN. The reagent reduces the binding of sVASN to its receptor CD71 and achieves the function of reducing the intracellular sVASN content, thereby limiting the development of cancer and achieving the purpose of treatment. In a specific embodiment, the inhibitor of CD71 has the function of blocking the binding of sVASN to its receptor CD71. Specifically, the binding site of sVASN to its receptor CD71 is at amino acids 1-85 of sVASN, that is, the action site of the reagent that blocks the binding of sVASN to its receptor CD71 is amino acids 1-85 of sVASN.

Specifically, the composition can be a pharmaceutical combination composition.

Preferably, the drug combination composition further comprises a pharmaceutically acceptable excipient, and the pharmaceutically acceptable excipient comprises any one or a combination of at least two of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a solubilizer, a solubilizing agent, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, an antibacterial agent or a buffer.

In another aspect, the present invention provides use of sVASN in promoting angiogenesis.

Preferably, the blood vessels are generated from vascular endothelial cells.

Specifically, the sVASN of the present invention has a sequence as shown in SEQ ID NO.1.

In another aspect, the present invention provides the use of sVASN in promoting cell proliferation and migration;

Preferably, the cells are cells that highly express CD71.

Preferably, the cells are ex vivo cells.

Preferably, the cells are commercially available cell lines.

Specifically, the sVASN refers to intracellular sVASN.

Specifically, the sVASN of the present invention has a sequence as shown in SEQ ID NO.1.

In another aspect, the present invention provides use of a reagent for detecting the content of sVASN in a cell in diagnosing cancer.

Preferably, the reagents for detecting the sVASN content in cells include reagents used in the following methods: hematoxylin-eosin staining (HE staining), safranin O-fast green staining, protein blotting (Western Blot method), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, immunohistochemistry (Immunohistochemistry) staining method, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytometry fluorescence resolution technology and protein chip method.

Preferably, the reagent for detecting the sVASN content in cells can also be a reagent used in the following methods: PCR-based detection method, Southern hybridization method, Northern hybridization method, dot hybridization method, fluorescence in situ hybridization method, DNA microarray method, ASO method, high-throughput sequencing platform method.

On the other hand, the present invention provides the use of CD71 in controlling the expression level of VASN.

Specifically, when the CD71 is overexpressed, the expression level of VASN increases; when the CD71 is inhibited, the expression level of VASN decreases.

More specifically, the VASN refers to endogenous VASN.

Preferably, the VASN expression level is protein expression level and mRNA expression level.

method

On the other hand, the present invention provides a method for inhibiting the entry of sVASN into cells (reducing the content/expression of sVASN in cells), which method comprises inhibiting the expression level and/or activity of CD71.

Specifically, the inhibition of CD71 expression and/or activity is achieved by administering a CD71 inhibitor to cells.

Preferably, the cells are ex vivo cells.

Preferably, the cells are commercially available cell lines.

Preferably, the cells may be of therapeutic or non-therapeutic interest.

On the other hand, the present invention provides a method for promoting sVASN to enter cells (increasing the content/expression level of sVASN in cells), which method comprises increasing the expression level and/or activity of CD71.

Specifically, the method includes the step of overexpressing CD71, such as constructing a vector containing the CD71 gene and transferring it into cells for expression.

Preferably, the cells are ex vivo cells.

Preferably, the cells are commercially available cell lines.

Preferably, the cells may be of therapeutic or non-therapeutic interest.

In another aspect, the present invention provides a method of promoting angiogenesis, the method comprising administering sVASN to a subject.

Specifically, the sVASN of the present invention has a sequence as shown in SEQ ID NO.1.

In another aspect, the present invention provides a method of treating cancer, the method comprising administering to a cancer patient a combination of one or more of the following agents:

1) CD71 inhibitors,

2) sVASN inhibitors, or

3) Agents that block the binding of sVASN to its receptor CD71.

Specifically, the route of administration can be systemic or local, oral or parenteral administration. The dosage is determined according to the drug used in the present invention, age, body weight, symptoms, therapeutic effect, route of administration, course of treatment, etc. As mentioned above, the dosage varies according to various conditions, so sometimes a dosage lower or higher than the above dosage can be used.

Preferably, the treatment method can also be used in combination with other cancer treatment methods.

Preferably, the treatment methods for other cancers include surgical therapy, chemotherapy, radiotherapy, gene therapy, immunotherapy, etc.

Specifically, the chemotherapy is to treat the patient with chemical drugs (chemo drugs), such as: thiotepa, cyclosphosphamide, difluoromethylornithine (DMFO), retinoic acid, piposulfan, dolastatin, busulfan, improsulfan, clodronate, camptothecin, bryostatin, capecitabine, carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, and any pharmaceutically acceptable salt, acid or derivative of any of the above.

Specifically, the radiotherapy includes ionizing radiation to the tumor, achieving extensive damage to DNA, DNA precursors, DNA replication and repair, chromosome assembly and maintenance. The ionizing radiation includes x-ray radiation, ultraviolet radiation, infrared radiation, gamma ray radiation or microwave radiation.

Specifically, the surgical treatment includes resection, which refers to the removal, excision and/or destruction of all or part of the cancerous tissue. Surgical treatment also includes laser surgery, cryosurgery, electrosurgery and microscope-controlled surgery, etc.

Specifically, immunotherapy refers to a treatment method that artificially enhances or suppresses the body's immune function to achieve the purpose of treating the disease in response to the body's low or hyperimmune state. Tumor immunotherapy aims to activate the human immune system and rely on its own immune function to kill cancer cells and tumor tissues. It generally relies on the use of immune effector cells and molecules to target and destroy cancer cells.

Specifically, the gene therapy refers to administering therapeutic polynucleotides to patients. Viral vectors for expressing polynucleotides are well known in the art and include eukaryotic expression systems such as adenovirus, adeno-associated virus, retrovirus, herpes virus, slow virus, poxvirus (including vaccinia virus) and papillomavirus (including SV40). Alternatively, the administration of polynucleotides can be with lipid-based carriers, such as liposomes.

In another aspect, the present invention provides a method for diagnosing cancer, comprising the step of detecting the content of sVASN in cancer cells.

Specifically, a high level of sVASN indicates a risk of cancer.

On the other hand, the present invention provides a method for screening drugs, which comprises contacting a candidate drug with cells that highly express CD71, detecting the cell migration and proliferation ability and activity rate before and after the contact, and the drug that reduces the cell migration and proliferation ability is a therapeutic drug for cancers that highly express CD71.

Preferably, the cells include vascular endothelial cells, colon cancer cells, glioma cells, endometrial cancer cells, keratinocytes, and embryonic kidney cells.

More preferably, the cells include human brain microvascular endothelial cells hEMEC/D3, human colon cancer cells HCT116, astroglioma cells U251, human endometrial cancer cells Ishikawa, human immortalized keratinocytes HaCaT, and human embryonic kidney cells HEK293.

In another aspect, the present invention provides a method for screening drugs, the method comprising contacting a candidate drug with vascular endothelial cells, detecting angiogenesis before and after the contact, and a drug that promotes angiogenesis is a pro-angiogenic drug.

Preferably, the cells include human brain microvascular endothelial cells hEMEC/D3.

General Concepts

As used herein, the term "inhibitor" refers to a substance that targets, reduces or inhibits at least one activity of a specific target gene or protein. In the present invention, it particularly refers to an active substance that targets, reduces or inhibits sVASN and/or CD71.

Preferably, the inhibitor includes those synthesized artificially or those existing in nature.

Preferably, the inhibitor includes an agent that reduces the expression of the target gene or protein through the following technologies: RNA interference technology (RNAi), antisense oligonucleotide (ASO) technology, CRISPR technology, TALEN technology, ZFN technology, Cre-loxP gene recombination technology.

Preferably, the inhibitor also includes a blocking peptide, an antibody, or a compound that specifically targets a target gene or protein.

Preferably, the CD71 inhibitor comprises an antibody targeting CD71.

Preferably, the CD71 inhibitor also includes Ferristatin.

The inhibitors described in the present invention particularly refer to substances that reduce the activity and/or expression of CD71, such as blocking peptides and Ferristatin in specific embodiments.

The term "antibody" as used herein includes glycosylated and non-glycosylated immunoglobulins of any isotype or subclass, or antigen binding regions thereof that compete with intact antibodies for specific binding, including human, humanized, chimeric, multispecific, monoclonal, polyclonal antibodies, and oligomers or antigen binding fragments thereof. It also includes proteins having antigen binding fragments or regions, such as Fab, Fab', F(ab')2, Fv, diabodies, Fd, dAb, large antibodies, single-chain antibody molecules, complementarity determining region (CDR) fragments, scFv, diabodies, triabodies, tetrabodies, and polypeptides, which contain at least a portion of an immunoglobulin sufficient to confer specific antigen binding to a target polypeptide.

As used herein, "cancer" and "tumor" are interchangeable terms that refer to any abnormal cell or tissue growth or proliferation in an animal. As used herein, the terms "cancer" and "tumor" encompass solid cancers and blood/lymphatic cancers, and also encompass malignant, pre-malignant, and benign growths, such as dysplasia. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific, non-limiting examples of such cancers include lung cancer, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), urethral cancer, squamous cell carcinoma, small cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, lung adenocarcinoma, lung squamous carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer (including uterine corpus endometrial cancer), salivary gland cancer, kidney cancer, renal cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, brain cancer, testicular cancer, cholangiocarcinoma, gallbladder cancer, gastric cancer, melanoma, mesothelioma, and various types of head and neck cancer.

Preferably, the cancer includes colon cancer, glioma (more specifically, astrocytic glioma cells), endometrial cancer, liver cancer, breast cancer, tongue squamous cell carcinoma.

More specifically, the cancers described herein include cancers of the head, neck, eye, mouth, larynx, esophagus, trachea, larynx, pharynx, chest, bone, lung, colon, rectum, stomach, prostate, bladder, uterus, cervix, breast, ovary, testis, skin, thyroid, blood, lymph node, kidney, liver, pancreas, brain or central nervous system.

Preferably, the cancer described in the present invention is a cancer with high expression of CD71.

The term "subject" as used herein refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., that is to be the recipient of a particular treatment. Generally, the terms "subject" and "patient" are used interchangeably herein when referring to human subjects.

"Treatment" as used herein means to alleviate or mitigate at least one symptom associated with such a disease, or to slow down or reverse the progression of such a disease. In the meaning of the present invention, the term "treatment" also means to inhibit, delay the onset of a disease (i.e., the period before the clinical manifestation of the disease) and/or reduce the risk of disease development or deterioration. For example, the term "treatment" related to cancer may refer to eliminating or reducing a patient's tumor load, or preventing, delaying or inhibiting metastasis, etc.

Specifically, the sVASN of the present invention has a sequence as shown in SEQ ID NO.1.

Detailed ways

The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes based on the technical solution and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram showing the results of mass spectrometry identification of CD71 as a VASN binding protein.

FIG2 shows the co-localization of endogenous VASN and CD71 in VASN high-expressing cells as determined by laser confocal microscopy.

FIG3 is a diagram showing the results of a co-immunoprecipitation experiment to determine the binding of sVASN to full-length CD71.

FIG. 4 is a diagram showing the results of molecular dynamics simulation to determine the region where sVASN binds to full-length CD71.

FIG5 is a diagram showing the results of an immunoprecipitation experiment to determine the binding of sVASN-dom1 to full-length CD71.

Figure 6 is a graph showing the results of an immunoprecipitation experiment to determine the binding of the sVASNC end to the full-length CD71.

FIG. 7 is a diagram showing the results of molecular dynamics simulation to determine the binding region between VASN truncation (1-240) and CD71.

Figure 8 is a graph showing the results of an immunoprecipitation experiment to determine the binding of sVASNC end 1-135 to full-length CD71.

Figure 9 is a graph showing the results of an immunoprecipitation experiment to determine that sVASNC end 120-240 does not bind to full-length CD71.

FIG. 10 is a graph showing the results of BIAcore assay of the affinity between VASN and CD71.

FIG. 11 is a graph showing the results of ELISA assay for the affinity of VASN to CD71.

FIG. 12 is a diagram showing the binding of sVASN, transferrin Tf and CD71 determined by molecular dynamics simulation.

Figure 13 is a graph showing the results of ELISA assay for competitive binding of transferrin, VASN and CD71, upper graph: transferrin competes for the binding of sVASN and CD71; lower graph: sVASN competes for the binding of transferrin and CD71.

FIG. 14 is a diagram showing the results of Western blot detection of exogenous sVASN entering cells.

FIG15 is a graph showing the results of Western blot detection showing that the amount of exogenous sVASN entering cells increases over time and decreases after blocking CD71.

FIG16 is a graph showing the results of Western blot detection showing that the amount of exogenous sVASN entering cells increases over time and decreases after CD71 inhibition.

FIG. 17 is a diagram showing the results of confirming that exogenous sVASN entered cells using laser confocal microscopy.

Figure 18 is a graph showing the results of laser confocal microscopy to determine the reduction in exogenous sVASN entering hEMEC/D3 cells after blocking CD71 or inhibiting CD71.

FIG. 19 is a graph showing the results of laser confocal microscopy to determine that the entry of exogenous sVASN into HaCaT cells was reduced after blocking CD71 or inhibiting CD71.

Figure 20 shows that exogenous sVASN enters cells to activate the YAP/TAZ signaling pathway, and blocking CD71 reduces the activation of the YAP/TAZ signaling pathway.

Figure 21 shows that exogenous sVASN enters cells to activate the YAP/TAZ signaling pathway, and inhibits CD71 to reduce the activation of the YAP/TAZ signaling pathway.

Figure 22 is a graph showing the results of sVASN promoting cell proliferation. sVASN has no effect on the CD71 low-expressing cell line U87 (the top is a graph showing the sVASN content in the culture supernatant of HepG2 detected by ELISA, and the bottom is a graph showing cell proliferation). Figure 23 is a graph showing the results of sVASN promoting cell migration. The migration-promoting effect of sVASN was weakened after treatment with CD71 blocking peptide.

FIG. 24 is a graph showing the results of sVASN promoting cell migration. The migration-promoting effect of sVASN was weakened after treatment with CD71 inhibitor.

FIG. 25 is a graph showing the results of sVASN promoting cell scratch healing. The effect of sVASN in promoting scratch healing was weakened after treatment with CD71 blocking peptide.

FIG. 26 is a graph showing the results of sVASN promoting cell scratch healing. The effect of sVASN in promoting scratch healing is weakened after treatment with CD71 inhibitor.

FIG. 27 is a graph showing the results of sVASN promoting angiogenesis. The angiogenesis-promoting effect of sVASN was weakened after treatment with CD71 blocking peptide.

General Methods

1. Immunoprecipitation experimental steps:

1. Cultivate cells using conventional methods, 10 cm dishes, transfect cells with 10 μg of plasmid (5 μg each for co-transfection), and use transfection reagents at a ratio of 1:2, using the same conventional techniques;

2. Extract total cell protein after 48 hours:

Rinse the cells once with pre-cooled PBS, add an appropriate amount of pre-cooled PBS, scrape the cells in the culture wells with a cell scraper on ice, transfer to an EP tube, and centrifuge at 3000rpm for 5min to collect the cells. Discard the supernatant, add 500μL of cell lysis buffer containing 1% protease inhibitors, and lyse the cells on ice for 50-60min. Centrifuge at 4℃, 12,000rpm for 10min, and transfer the supernatant to a new EP tube, which is the total cell protein.

3. Take 30-40 μL of total cell protein, add 10 μL of protein loading buffer, boil for 10 min, centrifuge at 12,000 rpm for 5 min, and use as input.

4. Add 30 μL of beads washed with co-IP buffer to the remaining total cell protein and rotate at 4°C for 1-2 hours to remove non-specifically bound proteins;

5. Add 3 μg of control antibody and rotate at 4°C for 2-3 hours;

6. Place the sample EP tube on a magnetic rack and let it stand for 5 minutes. Take the clear liquid along the side wall to a new EP tube, add 4 μg His or Flag antibody, and rotate at 4°C for 3 hours.

7. The IgG group was washed 3-4 times with co-IP buffer, and 30 μL of protein lysate and 7 μL of loading buffer were added, boiled for 10 min, and centrifuged at 12,000 rpm for 5 min. This is the IgG group;

8. Add 40 μL ProtienA/G magnetic beads to the EP tube of the antibody group and rotate at 4°C for 8 hours or overnight;

9. Incubate on a magnetic rack at 4°C for 5 min, remove most of the supernatant, wash 3-4 times with co-IP buffer, add 30 μL protein lysis buffer and 7 μL loading buffer, boil for 10 min, centrifuge at 12,000 rpm for 5 min, and perform SDS-PAGE electrophoresis;

10. Western blot analysis.

2. ELISA to determine affinity

1. Solution configuration:

ELISA coating solution (pH 9.6)

2. Coating: Coat the corresponding protein according to the purpose, dilute it to 10μg/mL with coating solution, and incubate at 4℃ overnight.

3. Washing: Wash once with PBST.

4. Binding: Add binding protein according to different concentration gradients, dilute with PBST, and incubate at 37°C for 2h.

5. Washing: Wash 3 times with PBST.

6. Add antibody: dilute at 1:200-1:500 and incubate at 37°C for 2 hours.

7. Washing: Wash 3 times with PBST.

8. Add secondary antibody: dilute at 1:10000 and incubate at 37℃ for 2h.

9. Washing: Wash 3 times with PBST.

10. Color development: TMB color development, incubate in dark for 10-15 minutes.

11. Stop: Add 50uL stop solution and immediately measure the OD value at 450nm.

3. Cell migration assay

1. Digest the cells with trypsin to form a single cell suspension and wash with PBS 2-3 times to remove residual serum;

2. Count, resuspend in serum-free medium, and count 2×10^5 cells per well;

3. Prepare sVASN at the same time and dilute it to the appropriate concentration with sterile water or PBS (Ferristatin to inhibit CD71 needs to be added 12-24 hours in advance, and CD71 blocking peptide needs to be added 6 hours in advance);

4. Carefully add the counted cells to the upper layer of the transwell chamber with a volume of 200 μL of cell culture medium and mix gently;

5. Add 800 μL of complete medium containing 10% serum to the lower chamber;

6. Continue to culture for 40 hours;

7. Aspirate the upper and lower culture media and fix with methanol for 20-30 minutes;

8. Add crystal violet dye and dye for 30 minutes;

9. Wash off the dye with clean water and carefully wipe off the inner layer of cells with absorbent cotton;

10. Carefully tear off the membrane or cut it with a scalpel, with the cell side (outside of the cell) facing up, and fix it with neutral gum;

11. Take photos and count under a microscope.

4. Cell scratch test

1. Cell plating: Digest the cells with trypsin to form a single cell suspension, count and spread on U-slide, and carefully remove the U-slide after the cells adhere to the wall after 24 hours;

2. Wash the cells with PBS, prepare sVASN, and dilute to the appropriate concentration with sterile water or PBS (Ferristatin inhibition of CD71 needs to be added 12-24 hours in advance, and CD71 blocking peptide needs to be added 6 hours in advance);

3. Add the calculated sVASN into the culture medium, with the volume of cell culture medium being 100 μL/chamber;

4. Continue to cultivate for the corresponding time;

5. Aspirate the culture medium and wash with PBS;

6. Take photos under a microscope and do statistics.

Example 1: Immunoprecipitation to determine the binding of VASN and CD71

In order to find the binding protein of VASN, we found through mass spectrometry that transferrin receptor CD71 is the main binding protein of VASN (Figure 1). In order to determine the binding sites of the two, we used the Discovery Studio protein-protein docking program (ZDOCK) to perform molecular docking of VASN and CD71. Since there is currently no resolved crystal structure of VASN, its structure was first predicted using AlphaFold. Since there are many flexible loops in the predicted structure of VASN, it may affect the docking results. At the same time, the experimental results confirmed the binding of soluble VASN (sVASN) to CD71, so the subsequent docking was performed using the extracellular segment of the 1-575 protein of VASN (sVASN) (as shown in SEQ ID NO.1). SEQ ID NO.1 is MCSRVPLLLPLLLLLALGPGVQGCPSGCQCSQPQTVFCTARQGTTVPRDVPPDTVGLYVFENGITMLDAGSFAGLPGLQLLDLSQNQIASLPSGVFQPLANLSNLDLTANRLHEITNETFRGLRRLERLYLGKNRIRHIQPGAFDTLDRLLELKLQDNELRALPPLRLPRLLLLDLSHNSLLALEPGILDTANVEALRLAGLGLQQLDEGLFSRLRNLHDLDVSDNQLERVPPVIRGLRGLTRLRLAGNTRIAQLRPEDLAGLAALQELDVSNLSLQALPGDLSG LFPRLRLLAAARNPFNCVCPLSWFGPWVRESHVTLASPEETRCHFPPKNAGRLLLELDYADFGCPATTTTATVPTTRPVVREPTALSSSLAPTWLSPTEPATEAPSPPSTAPPTVGPVPQPQDCPPSTCLNGGTCHLGTRHHLACLCPEGFTGLYCESQMGQGTRPSPTPVTPRPPRSLTLGIEPVSPTSLRVGLQRYLQGSSVQLRSLRLTYRNLSGPDKRLVTLRLPASLAEYTVTQLRPNATYSVCVMPLGPGRVPEGEEACGEAHTPPAVHSNHAPVTQAREGNLP.

Next, we constructed full-length vectors of sVASN-His and CD71-Flag and transfected HEK293T cells for co-immunoprecipitation experiments. The results showed that similar results were obtained regardless of whether Flag or His fishing was used, that is, sVASN and CD71 were bound (Figure 3).

Through molecular dynamics simulation, sVASN and CD71 complex can form a relatively stable complex conformation, and CD71 binds to the loop of sVASN. The interaction between CD71 and sVASN is mainly distributed in three regions: in the first region, GLY281 and GLU258 of sVASN form hydrogen bonds with ARG668 and THR666 of CD71; in the second region, ARG366 of sVASN forms hydrogen bonds with GLU149, LEU146, and TYR168 of CD71; in the third region, THR369, LEU371, and SER373 of sVASN form hydrogen bonds with SER142, ASP139, SER137, and GLU133 of CD71 (Figure 4).

Molecular docking results suggest that the amino acids 240-380 of sVASN form a loop to "lock" CD71. To determine whether the binding is affected by the loop, we constructed three sVASN truncations (sVASN-dom1: 1-240aa, sVASN-dom2: 240-380aa, sVASN-dom3: 380-575aa) and removed the loop to verify whether the binding exists. The results of co-immunoprecipitation showed that only sVASN-dom1 (1-240aa) bound to CD71 (Figure 5), indicating that the binding site is located in the 1-240 amino acid sequence.

In order to determine the binding of sVASN 1-240 amino acids to CD71, we selected the N-terminal and C-terminal truncations of 1-240aa (sVASN-Cdel: 1-220aa; sVASN-Ndel: 85-240aa) for co-immunoprecipitation experiments to determine the binding region. The results showed that sVASN-Cdel (1-220aa) could bind to CD71 (Figure 6), indicating that the binding site is located at the N-terminal (1-220aa).

To further identify the binding site, we used molecular dynamics simulation to determine the binding region between the sVASN truncate (1-240aa) and CD71. The results showed that the s’VASN truncate (1-240aa) can stably bind to the top domain of CD71 (Figure 7). The interaction between CD71 and sVASN (1-240aa) is mainly distributed as follows: CYS24, SER26, ILE64, TYR58, GLN79, ASP82, SER84, ARG135, ARG128 of sVASN form hydrogen bonds with ASN727, PHE726, THR729, ASN275, ASN251, GLY252, ILE272, ASN331, ARG325, PHE321 of CD71; GLU195, ASP220, GLY240, ARG198, SER224, GLN156 of sVASN form hydrogen bonds with LYS374, THR227, THR376, GLN197, SER378, ALA225, Asp194 of CD71 ( Figure 7 ).

Based on the results, we further constructed truncated forms of sVASN (1-240aa): sVASN-Vp1 (1-135aa) and sVASN-Vp2 (120-240aa). The results of co-immunoprecipitation showed that sVASN-Vp1 (1-135aa) bound to full-length CD71 (Figure 8), while sVASN-Vp2 (120-240aa) did not bind to full-length CD71 (Figure 9).

Based on the above results, we preliminarily determined that the binding site for CD71 is at amino acids 1-85 of sVASN ( Figures 5 , 6 , and 8 ).

Example 2: ELISA determination of affinity between VASN and CD71

Next, we measured the affinity of the two. First, by BIAcore, the results showed that the Kd value of sVASN binding to CD71 was 8.75nM (Figure 10).

At the same time, the traditional ELISA double sandwich method was used to determine the affinity under different coating conditions. The results showed that the sVASN coating binding Kd value was 153.5nM; the CD71 coating binding Kd value was 7.235nM (Figure 11), which was basically consistent with the BIAcore results, indicating that the binding affinity of the two was strong.

Next, we determined the binding sites of transferrin (TF), sVASN and CD71 through molecular dynamics simulation. The results showed that transferrin and sVASN bind to different sites of CD71, and there may be no competitive binding between the two (Figure 12). We further used the ELISA method to determine whether transferrin can compete for the binding of sVASN and CD71. CD71 was coated, and transferrin and sVASN were used for competition, respectively. The results showed that there was no competition between the two (Figure 13). This suggests that the site where TF binds to CD71 is different from the site where sVASN binds to CD71.

Example 3: Exogenous sVASN enters cells through CD71 endocytosis

The internalization of sVASN in different cells was further detected. Human brain microvascular endothelial cells hEMEC/D3, human colon cancer cells HCT116, astroglioma cells U251, human endometrial cancer cells Ishikawa, human immortalized keratinocytes HaCaT, and human embryonic kidney cells HEK293 were selected. These cells are known to have high levels of CD71 expression. By exogenously adding sVASN-His, cells at different time points were collected to detect the intracellular label protein level to determine the entry of exogenous sVASN into cells. The results showed that sVASN can enter cells, independent of cell type, and the amount of cells entering increases with time (Figures 14, 17).

In order to determine the role of CD71 in the internalization of exogenous sVASN into cells, we used CD71 blocking peptide (transferrin receptor peptide, abcam, ab101219) and CD71 inhibitor Ferristatin II (MCE, HY-D0256, which promotes the degradation of CD71 in vivo and in vitro) to detect the entry of exogenous sVASN into cells after CD71 blocking or inhibition.

The results showed that as time went on, the amount of exogenous sVASN entering the cells increased; when CD71 blocking peptide was added, the expression of CD71 remained almost unchanged, and the amount of exogenous sVASN entering the cells decreased; after the addition of CD71 inhibitor, the expression of CD71 decreased, which was consistent with the function of the inhibitor, and the amount of exogenous sVASN entering the cells decreased. The above results indicate that exogenous sVASN enters cells through CD71 (Figures 15, 16, 18, and 19).

Example 4: Biological functions of sVASN after it enters cells through CD71 receptor endocytosis

Next, we explored the biological function of sVASN after it enters the cell through CD71 receptor endocytosis. It is reported that VASN mainly exerts its biological functions such as promoting tumor proliferation through the YAP/TAZ signaling pathway, so we explored whether sVASN has a similar effect. After exogenous sVASN acts on different tumor cells, the YAP/TAZ signaling pathway is activated to varying degrees, and an sVASN dose-dependent relationship is presented (Figures 20-21). After blocking CD71 (Figure 20) or inhibiting CD71 (Figure 21), the activation degree of the YAP/TAZ signaling pathway is reduced, indicating that exogenous sVASN exerts its biological function through CD71.

Preliminary experiments determined that the sVASN content in the HepG2 culture supernatant was high, so we collected the HepG2 cell culture supernatant and performed ELISA to determine the sVASN concentration (Figure 22, top), and used the culture supernatant to detect its effect on cell proliferation. Normal cells and tumor cells were selected, and the proliferation of cells was determined by exogenously adding HepG2 cell culture supernatant. The results showed that, except for U87 with low CD71 expression, the HepG2 cell culture supernatant could significantly improve the proliferation ability of cells (Figure 22, bottom). The above results further confirmed that sVASN enters cells through CD71 receptor endocytosis, and sVASN has the ability to promote cell proliferation after entering cells.

We further selected human astrocytic glioblastoma U118 and U251 as research subjects to determine the effect of sVASN on glioma cells. The results showed that sVASN can promote the migration of U251 and U118 cells, and this promotion effect is weakened after blocking with CD71 blocking peptide (Figure 23). Similarly, the CD71 inhibitor Ferristatin also has a similar effect. After inhibiting CD71, the effect of sVASN in promoting the migration of glioma cells U251 and U118 is weakened (Figure 24).

The results of the cell scratch experiment were similar to those of the cell migration experiment. Exogenous sVASN could significantly enhance the healing ability of U251 and U118 cells, while this ability was weakened after blocking CD71 (Figure 25) or inhibiting CD71 (Figure 26), indicating that the effect of sVASN on cells was through the CD71 receptor.

Finally, we detected the effect of sVASN on angiogenesis. The angiogenesis kit (abcam, ab204726) was used to detect the angiogenesis ability of exogenous sVASN on human brain microvascular endothelial cells hEMEC/D3. The results showed that exogenous sVANS can significantly increase the number of vascular branch points and vascular length of hEMEC/D3, and increase with the increase of sVASN. After blocking CD71, the number of vascular branch points and vascular length decreased. This shows that the effect of sVASN on angiogenesis depends on the receptor CD71, that is, CD71 is a new functional receptor for sVASN.

The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims (10)

1. Use of any one of the following or a combination of any one or more of the following in the preparation of cancer drugs: 1) CD71 inhibitors, 2) sVASN inhibitors, or 3) agents that block the binding of sVASN to its receptor CD71; Specifically, the binding site of the sVASN to its receptor CD71 is at amino acids 1-85 of sVASN; Specifically, the reagent includes an inhibitor of CD71 and/or an inhibitor of sVASN; Preferably, the cancer comprises melanoma, fibrosarcoma, myxosarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat adenoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, Medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumors, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, gastric cancer, and forestomachal cancer; Preferably, the cancer is selected from colon cancer, glioma, endometrial cancer, liver cancer, breast cancer, tongue squamous cell carcinoma, pancreatic cancer, lung cancer, bladder cancer, lymphoma; Preferably, the cancer is a cancer with high expression of CD71; Preferably, the cancer is CD71-high expressing glioblastoma. 2. The use according to claim 1, wherein the inhibitor comprises an agent that reduces the expression of the target gene or protein by the following technologies: RNA interference technology, antisense oligonucleotide technology, CRISPR technology, TALEN technology, ZFN technology, Cre-loxP gene recombination technology; Preferably, the inhibitor also includes blocking peptides, antibodies, compounds that specifically target the target gene or protein; Preferably, the CD71 inhibitor includes antibodies and blocking peptides targeting CD71; Preferably, the CD71 inhibitor also includes Ferristatin. 3. The use according to claim 1, wherein the composition further contains other cancer drugs, the other cancer drugs comprising thiotepa, cyclophosphamide, difluoromethylornithine, retinoic acid, piposulfan, dolastatin, busulfan, improsulfan, clodronate, camptothecin, bryostatin, capecitabine, carboplatin, procarbazine, plicamycin, gemcitabine, navelbine, and any pharmaceutically acceptable salts, acids or derivatives thereof; Specifically, the composition is a drug combination composition; Preferably, the drug combination composition further comprises a pharmaceutically acceptable excipient, and the pharmaceutically acceptable excipient comprises any one or a combination of at least two of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a solubilizer, a solubilizing agent, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, an antibacterial agent or a buffer. 4. Application of sVASN in the preparation of drugs for promoting cell proliferation and migration or drugs for promoting angiogenesis; Preferably, the blood vessels are generated by endothelial cells; more preferably, vascular endothelial cells; Preferably, the cells are cells that highly express CD71; Preferably, the cell is a cancer cell with high expression of CD71; Specifically, the sVASN has a sequence as shown in SEQ ID NO.1. 5. A composition comprising at least two of the following: 1) CD71 inhibitors, 2) sVASN inhibitors, 3) an agent that blocks the binding of sVASN to its receptor CD71, or 4)sVASN; Specifically, the sVASN has a sequence as shown in SEQ ID NO.1; Specifically, the binding site of the sVASN to its receptor CD71 is at amino acids 1-85 of sVASN; Specifically, the reagent includes an inhibitor of CD71 and/or an inhibitor of sVASN; Preferably, the composition is a pharmaceutical combination composition; Preferably, the drug combination composition further comprises a pharmaceutically acceptable excipient, and the pharmaceutically acceptable excipient comprises any one or a combination of at least two of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an emulsifier, a solubilizer, a solubilizing agent, an osmotic pressure regulator, a surfactant, a coating material, a colorant, a pH regulator, an antioxidant, an antibacterial agent or a buffer. 6. Application of reagents for detecting sVASN content in cells in the diagnosis of cancer; Preferably, the reagents for detecting the sVASN content in cells include reagents used in the following methods: hematoxylin-eosin staining, safranin O-fast green staining, protein blotting, enzyme-linked immunosorbent assay, radioimmunoassay, sandwich assay, immunohistochemical staining method, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytometry fluorescence resolution technology and protein chip method; Preferably, the reagent for detecting the sVASN content in the cell is a reagent used in the following methods: a PCR-based detection method, a Southern hybridization method, a Northern hybridization method, a dot hybridization method, a fluorescence in situ hybridization method, a DNA microarray method, an ASO method, a high-throughput sequencing platform method; Specifically, the sVASN has a sequence as shown in SEQ ID NO.1; Preferably, the cancer is selected from colon cancer, glioma, endometrial cancer, liver cancer, breast cancer, tongue squamous cell carcinoma, pancreatic cancer, lung cancer, bladder cancer, lymphoma; Preferably, the cancer is a cancer with high expression of CD71. 7. Application of CD71 as a receptor for sVASN to control the expression of VASN; Specifically, when the CD71 is overexpressed, the expression level of VASN increases; when the CD71 is inhibited, the expression level of VASN decreases; Preferably, the VASN expression level is protein expression level and mRNA expression level. 8. A method for inhibiting sVASN entry into cells, the method comprising inhibiting the expression and/or activity of CD71; Specifically, the inhibition of CD71 expression and/or activity is achieved by administering a CD71 inhibitor to the cells; Preferably, the inhibitor includes an agent that reduces the expression of the target gene or protein by the following technologies: RNA interference technology, antisense oligonucleotide technology, CRISPR technology, TALEN technology, ZFN technology, Cre-loxP gene recombination technology; Preferably, the inhibitor also includes blocking peptides, antibodies, compounds that specifically target the target gene or protein; Preferably, the CD71 inhibitor includes antibodies and blocking peptides targeting CD71; Preferably, the CD71 inhibitor also includes Ferristatin. 9. A method for promoting sVASN to enter cells, the method comprising increasing the expression of CD71; Specifically, the method includes the step of overexpressing CD71. 10. A method for screening drugs, the method being selected from any one of the following: 1) The method comprises contacting a candidate drug with cells that highly express CD71, detecting the cell migration and proliferation ability and activity rate before and after the contact, and the drug that reduces the cell migration and proliferation ability is a therapeutic drug for cancers that highly express CD71; Preferably, the cells include vascular endothelial cells, colon cancer cells, glioma cells, endometrial cancer cells, keratinocytes, and embryonic kidney cells; More preferably, the cells include human brain microvascular endothelial cells hEMEC/D3, human colon cancer cells HCT116, astroglioma cells U251, human endometrial cancer cells Ishikawa, human immortalized keratinocytes HaCaT, and human embryonic kidney cells HEK293; 2) The method comprises contacting the candidate drug with vascular endothelial cells, detecting angiogenesis before and after the contact, and the drug that promotes angiogenesis is a pro-angiogenic drug; Preferably, the cells include human brain microvascular endothelial cells hEMEC/D3.